In the last five years our laboratory has identified the genetic basis of two dominantly inherited autoinflammatory disorders. The TNF receptor-associated periodic syndrome (TRAPS) is characterized by oftentimes prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. Because it was first recognized in a large family of Scottish-Irish ancestry, this condition had initially been called ?familial Hibernian fever.? In 1999 our group identified the first six mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in seven families of various ancestries with this clinical condition, including the original ?Hibernian? family, and therefore proposed the more ethnically neutral TRAPS nomenclature currently in use. In three members of a family with the C52F mutation, we found a defect in the activation-induced shedding of the p55 (but not the p75) TNF receptor, possibly leading to impaired homeostasis in the inflammatory response. Subsequently, our laboratory has identified additional TRAPS mutations, studied genotype-phenotype correlations, and established p55 receptor shedding defects for some, but not all, mutations tested. We also conducted an open-label, dose-escalation study that supported the efficacy of the TNFR p75:Fc fustion protein, etanercept, in TRAPS, and we developed TRAPS knockin mice for further mechanistic studies. In 2002 we and a French group independently discovered dominantly inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), that cause a distinct disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and arthropathy (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. In our initial cohort of 13 mutations, we identified mutations in 6, all in exon 3, which encodes the NACHT (or nucleotide binding site) domain of the cryopyrin protein. Consistent with role of cryopyrin in IL-1 regulation, we found evidence of increased monocyte IL-1beta by Western blot in a mutation-positive patient, compared with healthy controls. In subsequent studies we have identified additional CIAS1 mutations in NOMID/CINCA patients, but have not found mutations in several related proteins among CIAS1 mutation-negative patients. During the previous reporting period our collaborator, Dr. Raphaela Goldbach-Mansky, began treatment a treatment protocol for NOMID/CINCA with the IL-1 receptor antagonist, anakinra (IL-1RN), and for patients with familial cold autoinflammatory syndrome (FCAS) with another investigational IL-1 inhibitory agent. Results of the Last Year Gene expression studies in NOMID/CINCA: We have used Affymetrix U133A 2.0 microarrays to identify disease-specific gene expression profiles in the peripheral blood mononuclear cells of 12 NOMID/CINCA patients at baseline and during their treatment with anakinra. Among the genes that were at least 1.5-fold downregulated in patients after treatment at a significance level of less than or equal to 0.01 were several cytokine receptors (IL-1RN, IFNGR1, IFNGR2, IL6R, IL8R, IL10RB, IL17R, TNFRSF1A), genes involved in the immune response and regulation of the NF-kappa B pathway (CD59, IFITM2, DAF, NCF4, TLR1, TLR2, TLR5, TLR8, CR1, IKBKG, NKIRAS2), cell adhesion and chemotaxis (ICAMs, CCR1, CCR2, C3AR1, C5R1), regulation of apoptosis (CFLAR, DAP, BAX inhibitor, MCL1, BAG1), and leukotriene biosynthesis (ALOX5, ALOX5AP, ALOX12). Given that the downregulaton of these genes by anakinra correlates with marked clinical improvement, it is likely that some of these genes are involved in the pathogenesis of disease. Validation studies are in progress. Generation of new animal models: We are currently in the initial stages of developing a number of new animal models for the dominantly-inherited human diseases caused by cryopyrin mutations. These conditions range in severity from FCAS at the mild end of the spectrum, to Muckle-Wells syndrome at an intermediate level, to NOMID/CINCA at the severe end of the spectrum. We have now generated targeting vectors to ?knock in? 1 mutation each causing FCAS and MWS, and two different mutations causing different levels of severity of NOMID/CINCA. Electroporations are in progress, and we hope to have several mouse models of CIAS1 disease within the next year. Constructs have also been made to develop mouse models of yet another dominantly inherited autoinflammatory disease, the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA). This condition is characterized by severe acne, sterile abscesses, and sterile effusions of the joints. It is caused by mutations in PSTPIP1, a protein that binds pyrin, the familial Mediterranean fever protein. We have currently made two constructs to ?knock in? PAPA-associated mutations into the mouse germline, and a construct to ablate the expression of PSTPIP1 to create a knockout mouse. Conclusions and Significance Gene expression studies of patients with NOMID/CINCA confirm the central role of IL-1 signaling in the pathogenesis of this disorder. During the next year we will follow up on genes identified through microarray analysis, both in studies of pathophysiology and as candidates for screening in mutation-negative patients with clinical NOMID/CINCA. We also plan to begin in earnest studies of knockin mice described above, and to continue, in collaboration with Dr. Raphaela Goldbach-Mansky, therapeutic trials of IL-1 inhibition in CIAS1 disease. In addition, we hope to have PAPA knockin mice available, and have just re-initiated studies of TRAPS knockin mice.